Rice Embryo Globulins: Amino-Terminal Amino Acid Sequences, cDNA Cloning and Expression

A globulin fraction prepared from rice embryos contained polypeptidesor polypeptide groups of 49 kDa (designated REG1), 46 kDa (designatedREG2), about 35 kDa, 32 kDa and 25 kDa. The amino-terminal sequencesof REG1 and the major polypeptide in the 35-kDa group were identical,suggesting that the REG1 polypeptide undergoes partial proteolyticprocessing that removes a carboxy-terminal region. A cDNA clone,designated pcREG2, encoding REG2 was isolated, and its nucleotidesequence was determined. The deduced amino acid sequence ofREG2 was found to be 68% identical to that of the maize GLB2globulin. Reg2 mRNA was present at high levels during embryodevelopment for up to 14 days after flowering (DAF). Lower levelswere found 20 DAF when the maturation of embryos was almostcompleted, and at the dry mature stage. Reg2 mRNA almost disappearedupon imbibition of isolated dry mature embryos but it was re-inducedat a low level by further treatment with ABA. The expressionof Reg2 was not induced by ABA in suspension-cultured cells,unlike that of Osem, one of the late embryogenesis abundantprotein (LEA) genes. (Received November 6, 1995; Accepted April 22, 1996)     

Cloning and characterization of the EIN2-homology gene LeEIN2 from tomato.

A full-length cDNA, named LeEIN2 was cloned from tomato (Lycopersicon esculentum) by RT-PCR and RACE. Then the genomic DNA sequence of LeEIN2 was isolated by LA (Long and Accurate) PCR and Inverse PCR. This DNA sequence was 6838 bp including six introns. The LeEIN2 cDNA was 4343 bp and contained a 3951 bp open reading frame, encoding a 142.63 kDa protein of 1316 amino acids. Comparison of this protein sequence with that of Arabidopsis and Petunia showed that the amino acid homology was 66 and 90%, respectively. Northern blotting analysis indicated that the expression level of the LeEIN2 in young leaves was higher than in mature leaves and senescent leaves. During the development of fruits, the expression of the LeEIN2 was detected before mature green stage and got up to maximum at mature green and breaker stages, then reduced rapidly after breaker stage. The expression level of LeEIN2 in wild type tomato fruit at mature green stage did not distinctively change when treated with exogenous ethylene.     

Chloroplast and cytosolic glutamine synthetase are encoded by homologous nuclear genes which are differentially expressed in vivo

We have shown that the individual members of the plant gene family for glutamine synthetase (GS) are differentially expressed in vivo, and each encode distinct GS polypeptides which are targeted to different subcellular compartments (chloroplast or cytosol). At the polypeptide level, chloroplast GS (GS2) and cytosolic GS (GS1 and GSn) are distinct and show an organ-specific distribution. We have characterized full length cDNA clones encoding chloroplast or cytosolic GS of pea. In vitro translation products encoded by three different GS cDNA clones, correspond to the mature GS2, GS1, and GSn polypeptides present in vivo. pGS185 encodes a precursor to the chloroplast GS2 polypeptide as shown by in vitro chloroplast uptake experiments. The pGS185 translation product is imported into the chloroplast stroma and processed to a polypeptide which corresponds in size and charge to that of mature chloroplast stromal GS2 (44 kDa). The 49 amino terminal amino acids encoded by pGS185 are designated as a chloroplast transit peptide by functionality in vitro, and amino acid homology to other transit peptides. The cytosolic forms of GS (GS1 and GSn) are encoded by highly homologous but distinct mRNAs. pGS299 encodes the cytosolic GS1 polypeptide (38 kDa), while pGS341 (Tingey, S. V., Walker, E. L., and Coruzzi, G. M. (1987) EMBO. J. 6, 1-9) encodes a cytosolic GSn polypeptide (37 kDa). The homologous nuclear genes for chloroplast and cytosolic GS show different patterns of expression in vivo. GS2 expression in leaves is modulated by light, at the level of steady state mRNA and protein, while the expression of cytosolic GS is unaffected by light. The light-induced expression of GS2 is due at least in part to a phytochrome mediated response. Nucleotide sequence analysis indicates that chloroplast and cytosolic GS have evolved from a common ancestor and suggest a molecular mechanism for chloroplast evolution.     

The 23 KDa polypeptide of the photosynthetic oxygen-evolving complex from mustard seedlings (Sinapis alba L.). Nucleotide sequence of cDNA and evidence for phytochrome control of its mRNA abundance

The nucleotide sequence of a cDNA from mustard shows 78% homology in deduced amino acid sequence for the mature protein compared to the sequence for the 23 kDa protein of the oxygen-evolving complex from spinach [(1987) FEBS Lett. 216, 234-240]. There is also a high degree of homology between the premature protein sequences concerning the hydrophobic domain and its distance from the suggested processing site. The accumulation of mRNA for the 23 kDa protein in mustard was stimulated by continuous far-red light and reversal experiments by means of red/far-red light pulse treatment show the involvement of phytochrome in controlling the mRNA abundance for the 23 kDa polypeptide in mustard. The accumulation of the mRNA can be inhibited in white light if the seedlings are treated with the herbicide Norflurazon.     

Cloning, characterization and expression analysis of a sucrose synthase gene from tropical epiphytic orchid Oncidium Goldiana

A full-length cDNA encoding sucrose synthase was isolated from the tropical epiphytic orchid Oncidium Goldiana. The cDNA is 2829 bp in length containing an open reading frame of 2447 bp encoding 816 amino acids with a predicted molecular mass of 93.1 kDa. The deduced amino acid sequence of O . Goldiana sucrose synthase ( Osus ) shares more than 80% identity with those from other monocotyledonous plants. The sucrose synthase gene was demonstrated to encode a functional sucrose synthase protein by expression as recombinant protein in Escherichia coli . The Osus mRNA is present in all the tissues analysed, with the highest levels in strong sinks such as developing inflorescence and root tips. Incubation with sucrose or glucose resulted in a significant increase in the steady-state Osus mRNA levels in root tips and mature leaves in a similar pattern to maize Sus1 . Expression of the Osus mRNA in mature leaves was markedly enhanced by anaerobic conditions and elevated CO₂. The expression pattern and regulation of the gene suggest that the sucrose synthase plays an important role in the growth and development of the tropical epiphytic orchid O . Goldiana.     

光和糖对水稻Rubisco活化酶基因表达的影响

水稻黄化苗在光照2h内其Rubisco。活化酶的mRNA和蛋白量明显增加,然后维持在相对稳定的水平。光对水稻Rubisco活化酶的基因表达的诱导作用主要在转录水平上。Rubisco活化酶主要在绿叶中表达,这与Rubisco基因表达的器官特异性完全一致。用等渗葡萄糖喂养成熟的水稻叶片1h,促使水稻Rubisco大、小亚基和Rubisco活化酶可翻译mRNA含量下降。同样蔗糖对Rubisco小亚基和Rubisco活化酶的表达也有抑制,其作用弱于葡萄糖。     

Characterization of cDNA clones encoding the extrinsic 23 kDa polypeptide of the oxygen-evolving complex of photosystem II in pea

The 23 kDa polypeptide of the oxygen-evolving complex of photosystem II has been extracted from pea photosystem II particles by washing with 1 M NaCl and purified by anion-exchange chromatography. The N-terminal amino acid sequence has been determined and specific antisera have been raised in rabbits and used to screen a pea-leaf cDNA library in gt11. Determination of the nucleotide sequence of two clones provided the nucleotide sequence for the full 23 kDa polypeptide. The deduced amino acid sequence showed it to code for a mature protein of 186 amino acid residues with an N-terminal presequence of 73 amino acid residues showing a high degree of conservation with previously reported 23 kDa sequences from spinach and Chlamydomonas. Southern blots of genomic DNA from pea probed with the labelled cDNA gave rise to only one band suggesting that the protein is encoded by a single gene. Northern blots of RNA extracted from various organs indicated a message of approximately 1.1 kb, in good agreement with the size of the cDNA, in all chlorophyll-containing tissues. Western blots of protein extracted from the same organs indicated that the 23 kDa polypeptide was present in all major organs of the plant except the roots.Abbreviations bis-Tris bis (2-hydroxyethyl) imino-tris (hydroxymethyl)-methane - pfu plaque-forming units     

Characterization of a 46 kda insect chitinase from transgenic tobacco

A 46 kDa Manduca sexta (tobacco hornworm) chitinase was isolated from leaves of transgenic tobacco plants containing a recombinant insect chitinase cDNA, characterized, and tested for insecticidal activity. The enzyme was purified by ammonium sulfate fractionation, Q-Sepharose anion-exchange chromatography and mono-S cation-exchange chromatography. Although the gene for the chitinase encoded the 85 kDa full-length chitinase as previously reported by Kramer et al. [Insect Biochem. Molec. Biol. 23, 691–701 (1993)], the enzyme is produced in tobacco as a 46 kDa protein that is approximately four-fold less active than the 85 kDa chitinase. The N-terminal amino acid sequence of the 46 kDa chitinase is identical to that of the 85 kDa chitinase. The former enzyme is not glycosylated, whereas the latter contains approximately 25% carbohydrate. The pH and temperature optima of the 46 kDa chitinaseare similar to those of the 85 kDa chitinase. The former enzyme is more basic than the latter. The 46 kDa chitinase likely consists of the N-terminal catalytic domain of the 85 kDa chitinase and lacks the C-terminal domain that contains several potential sites for glycosylation. The 46 kDa chitinase is expressed in a number of plant organs, including leaves, flowers, stems and roots. Enzyme levels are higher in leaves and flowers than in stems and roots, and leaves from the middle portion of the plant have more chitinase than leaves from the top and bottom portions. Little or no enzyme is secreted outside of the plant cells because it remains in the intracellular space, even though its transit sequence is processed. When fed at a 2% dietary level, the 46 kDa chitinase caused 100% larval mortality of the merchant grain beetle, Oryzaephilis mercator. The results of this study support the hypothesis that insect chitinase is a biopesticidal protein for insect pests feeding on insect chitinase gene-containing transgenic plants.     

Accumulation of plastid HSP 23 of Chenopodium rubrum is controlled post-translationally by light

The expression of the 23 kDa plastid heat-shock protein (HSP) of Chenopodium rubrum has been studied at various light intensities at a temperature of 38°C where the 23 kDa protein accumulates to its highest levels. It was observed that the level of mRNA which is induced at this heat-shock temperature is independent of the light intensity between 0 and 1000 W m⁻². Labelling in vivo of all investigated HSP is also not dependent on the light fluxes applied. In clear contrast the accumulation of the mature chloroplast HSP 23 is light dependent: while almost no protein is detectable in the dark the level of the accumulated protein reaches a maximum at a light intensity of 300 W m⁻². The accumulated levels of HSP 23 correlate well with resistance against photoinhibition; photoinhibitory effects are observed at a light intensity of 300 W m⁻² or above as measured by the decline of PS II activity. When high light intensities are applied during recovery from heat shock the amounts of HSP 23 stay elevated for a longer time and at a higher level than at the standard light intensity of 10 W m⁻². This appears to be a peculiar property of the plastid HSP 23 as the accumulation of HSP 17 and 70, as analysed by Western blot, is not influenced by light. When under particular stress conditions the levels of HSP 23 remain low a protein of 31 kDa accumulates that reacts with the antibody to HSP 23 and might represent the precursor of HSP 23.